1. Field of the Invention
This invention relates to novel multibinding compounds that bind to sodium (Na+) channels and modulate their activity. The compounds of this invention comprise 2-10 Na+ channel ligands covalently connected by a linker or linkers, wherein the ligands in their monovalent (i.e., unlinked) state bind to and are capable of modulating the activity of one or more types of Na+ channel. The manner of linking the ligands together is such that the multibinding agents thus formed demonstrate an increased biologic and/or therapeutic effect as compared to the same number of unlinked ligands made available for binding to the Na+ channel. The invention also relates to methods of using such compounds and to methods of preparing them.
The compounds of this invention are particularly useful for treating diseases and conditions of mammals that are mediated by Na+ channels. Accordingly, this invention also relates to pharmaceutical compositions comprising a pharmaceutically acceptable excipient and an effective amount of a compound of this invention.
2. State of the Art
Voltage-gated ion channels play a critical role in shaping the electrical activity of neuronal and muscle cells, and in controlling the secretion of neurotransmitters and hormones through the gating of calcium ion entry. Large families of voltage-gated sodium (Na+), potassium (K+) and calcium (Ca2+) ion channels have been defined using electrophysiological, pharmacological and molecular techniques; they are named according to their selective permeability for a particular cation with reference to their voltage dependence, kinetic behavior or molecular identity.
Although the structures of Na+, K+ and Ca2+ channels are quite different, there are common functional elements represented in each. The channels are all transmembrane proteins with an ion-selective aqueous pore that, when open, extends across the membrane. Channel opening and closing (gating) is controlled by a voltage-sensitive region of the protein containing charged amino acids that move within the electric field. The movement of these charged groups leads to conformational changes in the structure of the channel resulting in conducting (open/activated) or nonconducting (closed/inactivated) states.
Voltage-gated Na+ channels mediate regenerative inward currents that are responsible for the initial depolarization of action potentials in brain neurons. Na+ channels are large glycoproteins that consist of various subunits, the principal one being the alpha (xcex1) subunit. Na+ channels exist as dimers in cardiac and skeletal muscles and exist as heterotrimers in neuronal cells. FIG. 1A shows that the xcex1 subunit has a modular architecture; it consists of four internally homologous domains (labeled I-IV), each of which contains six transmembrane segments. Prominant phosphorylation sites of the xcex1 subunit are also shown. The four domains fold together so as to create a central pore whose structural constituents determine the selectivity and conductance properties of the channel as shown in FIG. 1B. Auxiliary beta (xcex2) subunits are important modulators of Na+ channel function. Biochemical studies reveal the existence of two distinct xcex2 subunits (xcex21 and xcex22) associated with the brain Na+ channel. It should be understood that, for purposes of simplification, other subunits that may be involved in or required for transporter activity have been omitted from the diagrams.
Na+ channels can exist in multiple ion conducting (open) and nonconducting (closed/inactivated) conformations. FIG. 2A illustrates how Na+ channels open and then rapidly inactivate following voltage stimulation. Transitions between these states occurs in a voltage and time-dependent manner. The time course and voltage dependency of Na+-channel activity can be described by separate activation and inactivation gating processes. Activation takes place upon depolarization of the membrane (xcex94Vm) and the channel adopts an open pore conformation allowing Na+ influx. Inactivation processes then change the channel conformation to a nonconducting, non-activatable state. Repolarization returns the channels from inactivated to resting conformations. FIG. 2B illustrates how Na+ channel opening may be prolonged by toxin binding. Toxins such as veratridine and batrachotoxin are activators that can bind to channels in the open conformation and stabilize the channel in a modified conducting state. This in effect removes or slows down the inactivation process allowing ion flux to continue from minutes to hours. Conversely, toxins such as tetrodotoxin (TTX) are blockers that can bind to the channel in the inactivated conformations. One method of distinguishing different Na+ channels is whether they are TTX-sensitive or TTX-resistant. (See, for example, Denyer, et al., xe2x80x9cHTS Approaches to Voltage-Gated Ion Channel Drug Discoveryxe2x80x9d, DDT, 3, No. 7, 323-332 (1998); Whalley, et al. xe2x80x9cBasic Concepts in Cellular Cardiac Electrophysiology: Part II: Block of Ion Channels by Antiarrhythmic Drugsxe2x80x9d, PACE, 18, Part I, 1686-1704 (1995); Goodman and Gilban""s xe2x80x9cThe Pharmacological Basis of Therapeuticsxe2x80x9d McGraw-Hill, Ninth Ed. Ch. 35, 851-856; and Doggrell, et al., xe2x80x9cIon channel Modulators as Potential Positive Inotropic Compounds for Treatment of Heart Failurexe2x80x9d, Clinical and Experimental Pharmacology and Physiology, 21833-843, 1994.)
Sodium channel blockers/modulators are employed to alleviate various disease conditions including, but not limited to, epilepsy, pain, anaesthesia, neuroprotection, arrhythmia, and migraine. (See, for example, PCT Publication WO 96/20935, European Patent Application EP 0869119, PCT Publication WO 97/27169, U.S. Pat. No. 5,688,830, Hunter and Loughhead xe2x80x9cVoltage-Gated Sodium Channel Blockers for the Treatment of Chronic Painxe2x80x9d, Current Opinion in CPNS investigational Drugs, 1999, vol. 1, no. 1, 72-81 and Loughhead et al., xe2x80x9cSynthesis of Mexiletine Stereoisomers and Related Compounds via SNAr Nucleophilic Substitution of a Cr(CO)8-Complexed Aromatic Fluoridexe2x80x9d J. Org. Chem. 1999, 64, 3373-3375.) Antiepileptic agents, include, for example, phenytoin, carbamazepine, and lamotrigine. Phenytoin is the prototypic antiepileptic sodium channel blocker and is efficacious in treating partial and generalized tonic-clonic seizures in humans. One important property of phenytoin is that it is capable of preventing seizures without producing sedation. Thus, phenytoin was the first antiepileptic to approach the therapeutic ideal of inhibiting abnormal brain activity characteristic of seizures without appreciably interfering with normal brain activity.
Carbamazepine, an iminostilbene derivative of tricyclic antidepressants, exhibits a spectrum of anticonvulsant activity very similar to that of phenytoin. In humans, it is effective against partial and generalized tonic-clonic seizures, but not against absence seizures. Lamotrigine has been used for treating partial and generalized tonic-clonic seizure.
Topiramate is a sulfamate-substituted monosaccharide, with a phenytoin-like profile in the maximal electroshock and pentylenetetrazol tests. These studies have also shown that it can control seizures in some genetic epilepsy models, in amygdala-kindled rats and in animals with ischemia-induced epilepsy. Clinical studies have shown that topiramate is effective as an add-on drug for treating simple or complex partial seizures with or without secondary generalization, even when administrered as monotherapy.
The clinical shortcomings of drugs in current usage are considerable. For example, lamotrigine causes rash and sedation and topiramate, phenytoin, and carbamazepine causes central nervous system side effects.
Thus, there continues to exist a need for novel compounds having improved therapeutic activities (e.g., increased potency, greater tissue selectivity, increased efficacy, reduced side effects and a more favorable duration of action.)
This invention is directed to novel multibinding compounds that bind to Na+ channels in mammalian tissues and can be used to treat diseases and conditions mediated by such channels.
Accordingly, in one of its composition aspects, this invention is directed to a multibinding compound and salts thereof comprising 2 to 10 ligands which may be the same or different and which are covalently attached to a linker or linkers, which may be the same or different, each of said ligands comprising a ligand domain capable of binding to a Na+ channel.
The multibinding compounds of this invention are preferably represented by formula I:
(L)p(X)qxe2x80x83xe2x80x83(I)
where each L is a ligand that may be the same or different at each occurrence; X is a linker that may be the same or different at each occurrence; p is an integer of from 2 to 10; and q is an integer of from 1 to 20; wherein each of said ligands comprises a ligand domain capable of binding to a Na+ channel. Preferably q is less than p.
Preferably, the binding of the multibinding compound to a Na+ channel or channels in a mammal modulates diseases and conditions mediated by the Na+ channel or channels.
In another of its composition aspects, this invention is directed to a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a therapeutically effective amount of one or more multibinding compounds (or pharmaceutically acceptable salts thereof) comprising 2 to 10 ligands which may be the same or different and which are covalently attached to a linker or linkers, which may be the same or different, each of said ligands comprising a ligand domain capable of binding to a Na+ channel of a cell mediating mammalian diseases or conditions, thereby modulating the diseases or conditions.
In still another of its composition aspects, this invention is directed to a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a therapeutically effective amount of one or more multibinding compounds represented by formula I,
(L)p(X)qxe2x80x83xe2x80x83(I)
or pharmaceutically acceptable salts thereof, where each L is a ligand that may be the same or different at each occurrence; X is a linker that may be the same or different at each occurrence; p is an integer of from 2 to 10; and q is an integer of from 1 to 20; wherein each of said ligands comprises a ligand domain capable of binding to a Na+ channel of a cell mediating mammalian diseases or conditions, thereby modulating the diseases or conditions. Preferably q is less than p.
In one of its method aspects this invention is directed to a method for modulating the activity of a Na+ channel in a biologic tissue, which method comprises contacting a tissue having a Na+ channel with a multibinding compound (or pharmaceutically acceptable salts thereof) under conditions sufficient to produce a change in the activity of the channel in said tissue, wherein the multibinding compound comprises 2 to 10 ligands which may be the same or different and which are covalently attached to a linker or linkers, which may be the same or different, each of said ligands comprising a ligand domain capable of binding to a Na+ channel.
In another of its method aspects, this invention is directed to a method for treating a disease or condition in a mammal resulting from an activity of a Na+ channel, which method comprises administering to said mammal a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable excipient and one or more multibinding compounds (or pharmaceutically acceptable salts thereof) comprising 2 to 10 ligands which may be the same or different and which are covalently attached to a linker or linkers, which may be the same or different, each of said ligands comprising a ligand domain capable of binding to a Na+ channel of a cell mediating mammalian diseases or conditions.
In yet another of its method aspects, this invention is directed to a method for treating a disease or condition in a mammal resulting from an activity of a Na+ channel, which method comprises administering to said mammal a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable excipient and one or more multibinding compounds represented by formula I,
(L)p(X)qxe2x80x83xe2x80x83(I)
and pharmaceutically acceptable salts thereof, where each L is a ligand that may be the same or different at each occurrence; X is a linker that may be the same or different at each occurrence; p is an integer of from 2 to 10; and q is an integer of from 1 to 20; wherein each of said ligands comprises a ligand domain capable of binding to a Na+ channel of a cell mediating mammalian diseases or conditions. Preferably q is less than p.
In a further aspect, this invention provides processes for preparing the multibinding agents of Formula I.
This invention is further directed to general synthetic methods for generating large libraries of diverse multimeric compounds which multimeric compounds are candidates for possessing multibinding properties. The diverse multimeric compound libraries provided by this invention are synthesized by combining a linker or linkers with a ligand or ligands to provide for a library of multimeric compounds wherein the linker and ligand each have complementary functional groups permitting covalent linkage. The library of linkers is preferably selected to have diverse properties such as valency, linker length, linker geometry and rigidity, hydrophilicity or hydrophobicity, amphiphilicity, acidity, basicity and polarization. The library of ligands is preferably selected to have diverse attachment points on the same ligand, different functional groups at the same site of otherwise the same ligand, and the like.
This invention is also directed to libraries of diverse emultimeric compounds which multimeric compounds are candidates for possessing multibinding properties. These libraries are prepared via the methods described above and permit the rapid and efficient evaluation of what molecular constraints impart multibinding properties to a ligand or a class of ligands targeting a receptor.
Accordingly, in one of its method aspects, this invention is directed to a method for identifying multimeric ligand compounds possessing multibinding properties which method comprises:
(a) identifying a ligand or a mixture of ligands wherein each ligand contains at least one reactive functionality;
(b) identifying a library of linkers wherein each linker in said library comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand;
(c) preparing a multimeic ligand compound library by combining at least two stoichiometric equivalents of the ligand or mixture of ligands identified in (a) with the library of linkers identified in (b) under conditions wherein the complementary functional groups react to form a covalent linkage between said linker and at least two of said ligands; and
(d) assaying the multimeric ligand compounds produced in (c) above to identify multimeric ligand compounds possessing multibinding properties.
In another of its method aspects, this invention is directed to a method for identifying multimeric ligand compounds possessing multibinding properties which method comprises:
(a) identifying a library of ligands wherein each ligand contains at least one reactive functionality;
(b) identifying a linker or mixture of linkers wherein each linker comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand;
(c) preparing a multimeric ligand compound library by combining at least two stoichiometric equivalents of the library of ligands identified in (a) with the linker or mixture of linkers identified in (b) under conditions wherein the complementary functional groups react to form a covalent linkage between said linker and at least two of said ligands; and
(d) assaying the multimeric ligand compounds produced in (c) above to identify multimeric ligand compounds possessing multibinding properties.
The preparation of the multimeric ligand compound library is achieved by either the sequential or concurrent combination of the two or more stoichiometric equivalents of the ligands identified in (a) with the linkers identified in (b). Sequential addition is preferred when a mixture of different ligands is employed to ensure heterodimeric or multimeric compounds are prepared. Concurrent addition of the ligands occurs when at least a portion of the multimer compounds prepared are homomultimeric compounds.
The assay protocols recited in (d) can be conducted on the multimeric ligand compound library produced in (c) above, or preferably, each member of the library is isolated by preparative liquid chromatography mass spectrometry (LCMS).
In one of its composition aspects, this invention is directed to a library of multimeric ligand compounds which may possess multivalent properties which library is prepared by the method comprising:
(a) identifying a ligand or a mixture of ligands wherein each ligand contains at least one reactive functionality;
(b) identifying a library of linkers wherein each linker in said library comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand; and
(c) preparing a multimeric ligand compound library by combining at least two stoichiometric equivalents of the ligand or mixture of ligands identified in (a) with the library of linkers identified in (b) under conditions wherein the complementary functional groups react to form a covalent linkage between said linker and at least two of said ligands.
In another of its composition aspects, this invention is directed to a library of multimeric ligand compounds which may possess multivalent properties which library is prepared by the method comprising:
(a) identifying a library of ligands wherein each ligand contains at least one reactive functionality;
(b) identifying a linker or mixture of linkers wherein each linker comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand; and
(c) preparing a multimeric ligand compound library by combining at least two stoichiometric equivalents of the library of ligands identified in (a) with the linker or mixture of linkers identified in (b) under conditions wherein the complementary functional groups react to form a covalent linkage between said linker and at least two of said ligands.
In a preferred embodiment, the library of linkers employed in either the methods or the library aspects of this invention is selected from the group comprising flexible linkers, rigid linkers, hydrophobic linkers, hydrophilic linkers, linkers of different geometry, acidic linkers, basic linkers, linkers of different polarization and amphiphilic linkers. For example, in one embodiment, each of the linkers in the linker library may comprise linkers of different chain length and/or having different complementary reactive groups. Such linker lengths can preferably range from about 2 to 100 xc3x85.
In another preferred embodiment, the ligand or mixture of ligands is selected to have reactive functionality at different sites on said ligands in order to provide for a range of orientations of said ligand on said multimeric ligand compounds. Such reactive functionality includes, by way of example, carboxylic acids, carboxylic acid halides, carboxyl esters, amines, halides, isocyanates, vinyl unsaturation, ketones, aldehydes, thiols, alcohols, anhydrides, and precursors thereof. It is understood, of course, that the reactive functionality on the ligand is selected to be complementary to at least one of the reactive groups on the linker so that a covalent linkage can be formed between the linker and the ligand.
In other embodiments, the multimeric ligand compound is homomeric (i.e., each of the ligands is the same, although it may be attached at different points) or heterodimeric (i.e., at least one of the ligands is different from the other ligands).
In addition to the combinatorial methods described herein, this invention provides for an interative process for rationally evaluating what molecular constraints impart multibinding properties to a class of multimeric compounds or ligands targeting a receptor. Specifically, this method aspect is directed to a method for identifying multimeric ligand compounds possessing multibinding properties which method comprises:
(a) preparing a first collection or iteration of multimeric compounds which is prepared by contacting at least two stoichiometric-equivalents of the ligand or mixture of ligands which target a receptor with a linker or mixture of linkers wherein said ligand or mixture of ligands comprises at least one reactive functionality and said linker or mixture of linkers comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand wherein said contacting is conducted under conditions wherein the complementary functional groups react to form a covalent linkage between said linker and at least two of said ligands;
(b) assaying said first collection or iteration of multimeric compounds to assess which if any of said multimeric compounds possess multibinding properties;
(c) repeating the process of (a) and (b) above until at least one multimeric compound is found to possess multibinding properties;
(d) evaluating what molecular constraints imparted multibinding properties to the multimeric compound or compounds found in the first iteration recited in (a)-(c) above;
(e) creating a second collection or iteration of multimeric compounds which elaborates upon the particular molecular constraints imparting multibinding properties to the multimeric compound or compounds found in said first iteration;
(f) evaluating what molecular constraints imparted enhanced multibinding properties to the multimeric compound or compounds found in the second collection or iteration recited in (e) above;
(g) optionally repeating steps (e) and (f) to further elaborate upon said molecular constraints.
Preferably, steps (e) and (f) are repeated at least two times, more preferably at from 2-50 times, even more preferably from 3 to 50 times, and still more preferably at least 5-50 times.